Screening And Application Of Nucleic Acid Aptamers For Clothianidin And Ethyl Carbamat

Small molecule hazards can enter organisms through the food chain and eventually pose a threat to human health,even causing cancer and death in severe cases.Therefore,it is of great significance to carry out the recognition and detection of small molecule hazards.The existing small molecule recognition molecules are mainly antibodies,which have problems such as easy inactivation,high cost and long production cycle.As a new type of biological recognition molecule,aptamer has the advantages of good stability,low cost and short production cycle,but the screening of aptamers for small molecule hazards such as clothianidin has not been reported.This study improved the Capture-SELEX technology established by our research group,screened the aptamers of clothianidin and ethyl carbamate,and revealed their binding mechanism to the target.Finally,the aptamer of clothianidin was used as a biometric molecule to create a new fluorescence detection method of clothianidin.The main contents of the study are as follows:（1）The Capture-SELEX technique was used to screen and recognize the aptamers of clothianidin.The library with the capacity of 10¹³-10¹⁵ ss DNA sequences was fixed on the surface of magnetic beads.After 10 rounds of screening,a 68-base CLO-1aptamer was successfully obtained,and its dissociation constant（K_d value）was40.66±3.47 n M.Circular dichroism experiments found that CLO-1 aptamer can be specifically recognized with clothianidin by folding into a hairpin structure.The results of molecular docking showed that clothianidin was bound in the hydrophobic cavity of CLO-1 aptamer through hydrophobic action,and was surrounded by side chain bases C-2,C-3,G-4,A-5,T-26,C-27,T-52,A-53,T-54,where C-2,C-3,A-53 bases formed stable hydrogen bonds with clothianidin.This study revealed that clothianidin molecules and CLO-1 aptamers bind stably mainly through hydrophobic action and hydrogen bonding force.（2）The Capture-SELEX technique was used to screen and recognize the aptamers of ethyl carbamate.The Capture-SELEX technique was further improved to enhance the affinity of the ethyl carbamate aptamer by preparing affinity libraries using Lambda exonuclease instead of magnetic beads.After 14 rounds of positive and counter screening,the aptamer of ethyl carbamate（named EC-1）was obtained.Subsequently,the sequence was truncated by molecular docking and other means,and it was found that ethyl carbamate was bound to the hydrophobic cavity of the truncated aptamer（named EC1-34）through hydrophobic interaction,and was surrounded by bases G-21,G-22 and G-23 on the stem loop,among which G-22 and G-23 groups formed hydrogen bonds with ethyl carbamate.The EC1-34 aptamer was found to bind stably to the ethyl carbamate molecule by hydrophobic interaction and hydrogen bond force.（3）A fluorescent aptasensor of clothianidin was constructed.Using the screened CLO-1 aptamer as the recognition molecule of clothianidin and Gene Green fluorescent dye as the sensing signal,a highly sensitive and selective clothianidin fluorescence detection sensor was constructed,and its minimum detection limit was 5.527 mg·L^-1,which can be applied to the detection of clothianidin residues in actual agricultural products.In conclusion,this study further improved the Capture-SELEX technology to obtain the aptamers against clothianidin and ethyl carbamate,and revealed that the binding of aptamer sequences to target molecules is closely related to hydrophobic interaction and hydrogen bonding,which provides a new idea for the biometric molecules and detection of small molecule hazards.